Mystery of Bacterial Growth and Resistance Solved: Findings Shed Light On How Bacteria Form Protective Biofilms

Scientists at The Scripps Research Institute have unraveled a complex chemical pathway that enables bacteria to form clusters called biofilms. Such improved understanding might eventually aid the development of new treatments targeting biofilms, which are involved in a wide variety of human infections and help bacteria resist antibiotics.

int on April 26, 2012, by the journal Molecular Cell, explains how nitric oxide, a signaling molecule involved in the immune system, leads to biofilm formation.

"It is estimated that about 80 percent of human pathogens form biofilms during some part of their life cycle," said Scripps Research president and CEO Michael Marletta, PhD, who led the work. "In this study, we have detailed for the first time the signaling pathway from nitric oxide to the sensor through cellular regulators and on to the biological output, biofilm formation."

"There's a lot of interest right now in finding ways to influence biofilm formation in bacteria," said lead author Lars Plate, a graduate student in Marletta's team, which recently moved to Scripps Research from the University of California, Berkeley. "Figuring out the signaling pathway is a prerequisite for that."

Biofilm formation is a critical phenomenon that occurs when bacterial cells adhere to each other and to surfaces, at times as part of their growth stage and at other times to gird against attack. In such aggregations, cells on the outside of a biofilm might still be susceptible to natural or pharmaceutical antibiotics, but the interior cells are relatively protected. This can make them difficult to kill using conventional treatments.

Biofilms can form on surgical instruments such as heart valves or catheters, leading to potentially deadly infections. Likewise, difficult-to-eliminate biofilms also play key roles in a host of conditions from gum disease to cholera, and from cystic fibrosis to Legionnaires' disease.

For years, the Marletta lab and other groups have been studying how nitric oxide regulates everything from blood vessel dilation to nerve signals in humans and other vertebrates. Past research had also revealed that nitric oxide is involved in influencing bacterial biofilm formation.

Nitric oxide in sufficient quantity is toxic to bacteria, so it's logical that nitric oxide would trigger bacteria to enter the safety huddle of a biofilm. But nobody knew precisely how.

In the new study, the scientists set out to find what happens after the nitric oxide trigger is pulled. "The whole project was really a detective story in a way," said Plate.

In vertebrates, nitric oxide can bind to something called the Heme-Nitric Oxide/Oxygen (H-NOX) binding domain on a specific enzyme, activating that enzyme and beginning the chemical cascades that lead to physiological functions such as blood vessel dilation.

Many bacteria also have H-NOX domains, including key pathogens, so this seemed the best starting point for the investigation. From there, the team turned to genomic data.

Genes for proteins that interact are often found adjacent to one another. Based on this fact, the researchers were able to infer a connection between the bacterial H-NOX domain and an enzyme called histidine kinase, which transfers phosphate chemical groups to other molecules in signaling pathways. The question was where the phosphates were going.

To learn more, the researchers used a technique called phosphotransfer profiling. This involved activating the histidine kinase and then allowing them to react separately with about 20 potential targets. Those targets that the histidine kinase rapidly transferred phosphates to had to be part of the signaling pathway. "It's a neat method that we used to get an answer that was in fact very surprising," said Plate.

The experiments revealed that the histidine kinase phosphorylated three proteins called response regulators that work together to control biofilm formation for the project's primary study species, the bacterium Shewanella oneidensis, which is found in lake sediments.

Further work showed that each regulator plays a complementary role, making for an unusually complex system. One regulator activates gene expression, another controls the activity of an enzyme producing cyclic diguanosine monophosphate, an important bacterial messenger molecule that is critical in biofilm formation, and the third tunes the degree of activity of the second.

Since other bacterial species use the same chemical pathway uncovered in this study, the findings pave the way to further explore the potential for pharmaceutical application. As one example, researchers might be able to block biofilm formation with chemicals that interrupt the activity of one of the components of this nitric oxide cascade.

Marletta's group has already explored nitric oxide's role in controlling Legionnaires' disease and, among other goals, will focus now on understanding biofilm formation in the bacterium that causes cholera.

This research was supported by the National Institutes of Health and a Chang-Lin Tien Graduate Fellowship in the Environmental Sciences.

Journal Reference:

1. Lars Plate, Michael A. Marletta. Nitric Oxide Modulates Bacterial Biofilm Formation through a Multicomponent Cyclic-di-GMP Signaling Network. Molecular Cell, 2012; DOI: 10.1016/j.molcel.2012.03.023

Courtesy: ScienceDaily

Two Drugs Better Than One to Treat Youth With Type 2 Diabetes, Study Suggests

Programs to prevent or delay type 2 diabetes in high-risk adults would result in fewer people developing diabetes and lower health care costs over time, researchers conclude in a new study funded by the National Institutes of Health.

Prevention programs that apply interventions tested in the landmark Diabetes Prevention Program (DPP) clinical trial would also improve quality of life for people who would otherwise develop type 2 diabetes. The analysis of costs and outcomes in the DPP and its follow-up study is published in the April 2012 issue of Diabetes Care and online March 22.

The DPP showed that lifestyle changes (reduced fat and calories in the diet and increased physical activity) leading to modest weight loss reduced the rate of type 2 diabetes in high-risk adults by 58 percent, compared with placebo. Metformin reduced diabetes by 31 percent. These initial results were published in 2002. As researchers monitored participants for seven more years in the DPP Outcomes Study (DPPOS), they continued to see lower rates of diabetes in the lifestyle and metformin groups compared with placebo. Lifestyle changes were especially beneficial for people age 60 and older.

The economic analysis of the DPP/DPPOS found that metformin treatment led to a small savings in health care costs over 10 years, compared with placebo. (At present, metformin, an oral drug used to treat type 2 diabetes, is not approved by the Food and Drug Administration for diabetes prevention.) The lifestyle intervention as applied in the study was cost-effective, or justified by the benefits of diabetes prevention and improved health over 10 years, compared with placebo.

"Over 10 years, the lifestyle and metformin interventions resulted in health benefits and reduced the costs of inpatient and outpatient care and prescriptions, compared with placebo. From the perspective of the health care payer, these approaches make economic sense," said the study's lead author William H. Herman, M.D., M.P.H., a co-investigator of the DPP Research Group and director of the Michigan Center for Diabetes Translational Research, Ann Arbor.

The DPP enrolled 3,234 overweight or obese adults with blood sugar levels higher than normal but below the threshold for diabetes diagnosis. Participants were randomly assigned to a lifestyle intervention aimed at a 7 percent weight loss and 150 minutes per week of moderate intensity activity, metformin treatment, or placebo pills. The groups taking metformin or placebo pills also received standard lifestyle recommendations.

"We don't often see new therapies that are more effective and at the same time less costly than usual care, as was the case with metformin in the DPP. And while the lifestyle intervention was cost-effective, we would see greater savings if the program were implemented in communities," said Griffin P. Rodgers, M.D., director of the NIH's National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). "This has already been demonstrated in other NIDDK-funded projects, including one in YMCAs, where a lifestyle-change program cost $300 per person per year in a group setting, compared to about $1,400 for one-on-one attention in the DPP."

In the DPP, direct costs over 10 years per participant for the lifestyle and metformin interventions were higher than for placebo ($4,601 lifestyle, $2,300 metformin, and $769 placebo). The higher cost of the lifestyle intervention was due largely to the individualized training those participants received in a 16-session curriculum during the DPP and in group sessions during the DPPOS to reinforce behavior changes.

However, the costs of medical care received outside the DPP, for example hospitalizations and outpatient visits, were higher for the placebo group ($27,468) compared with lifestyle ($24,563) or metformin ($25,616). Over 10 years, the combined costs of the interventions and medical care outside the study were lowest for metformin ($27,915) and higher for lifestyle ($29,164) compared with placebo ($28,236). Throughout the study, quality of life as measured by mobility, level of pain, emotional outlook and other indicators was consistently better for the lifestyle group.

"The DPP demonstrated that the diabetes epidemic, with more than 1.9 million new cases per year in the United States, can be curtailed. We now show that these interventions also represent good value for the money," said study chair David M. Nathan, M.D., director of the Diabetes Research Center at Massachusetts General Hospital, Boston.

The above story is reprinted from materials provided by NIH/National Institute of Diabetes and Digestive and Kidney Diseases.  

Courtesy: ScienceDaily

Mechanism That Could Contribute to Problems in Alzheimer's Identified

Scientists at the Gladstone Institutes have unraveled a process by which depletion of a specific protein in the brain contributes to the memory problems associated with Alzheimer's disease. These findings provide insights into the disease's development and may lead to new therapies that could benefit the millions of people worldwide suffering from Alzheimer's and other devastating neurological disorders.

The study, led by Gladstone Investigator Jorge J. Palop, PhD, revealed that low levels of a protein, called Nav1.1, disrupt the electrical activity between brain cells. Such activity is crucial for healthy brain function and memory. Indeed, the researchers found that restoring Nav1.1 levels in mice that were genetically modified to mimic key aspects of Alzheimer's disease (AD-mice) improved learning and memory functions and increased their lifespan. Their findings are featured on the cover of the April 27 issue of Cell, available online April 26.

"It is estimated that more than 30 million people worldwide suffer from Alzheimer's disease and that number is expected to rise dramatically in the near future," said Lennart Mucke, MD, who directs neurological research at Gladstone, an independent and nonprofit biomedical-research organization. "This research improves our understanding of the biological processes that underlie cognitive dysfunction in this disease and could open the door for new therapeutic interventions."

The researchers' findings suggest that Nav1.1 levels in special regulatory nerve cells called parvalbumin cells, or PV cells, are essential to generate healthy brain-wave activity -- and that problems in this process contribute to cognitive decline in AD-mice and possibly in patients with Alzheimer's.

In the brain, neurons form highly interconnected networks, using chemical and electrical signals to communicate with each other. The researchers investigated whether this communication between neurons is disrupted in AD-mice, and if so, how this may affect the symptoms of Alzheimer's disease.

To study this, they performed electroencephalogram (EEG) recordings -- a technique that detects abnormalities in the brain's electrical waves such as those found in patients with epilepsy. They found that similar abnormalities emerged during periods of reduced gamma-wave oscillations -- a type of brain wave that is crucial to regulating learning and memory.

"Like a conductor in an orchestra, PV cells regulate brain rhythms by precisely controlling excitatory brain activity," said Laure Verret, PhD, postdoctoral fellow and lead author. "We found that PV cells in patients with Alzheimer's and in AD-mice have low levels of the protein Nav1.1 -- likely contributing to PV cell dysfunction. As a consequence, AD-mice had abnormal brain rhythms. By restoring Nav1.1 levels, we were able to re-establish normal brain function."

Indeed, the scientists found that increasing Nav1.1 levels in PV cells improves brain wave activity, learning, memory and survival rates in AD-mice.

"Enhancing Nav1.1 activity, and consequently improving PV cell function, may help in the treatment of Alzheimer's disease and other neurological disorders associated with gamma-wave alterations and cognitive impairments such as epilepsy, autism and schizophrenia," said Dr. Palop, who is also an assistant professor of neurology at the University of California, San Francisco, with which Gladstone is affiliated. "These findings may allow us to develop therapies to help patients with these devastating diseases."

Other scientists who participated in this research at Gladstone include Giao Hang, PhD, Kaitlyn Ho, Nino Devidze, PhD, and Anatol Kreitzer, PhD. Funding was provided by a variety of sources, including the National Institutes of Health, the Stephen D. Bechtel, Jr. Foundation, the Philippe Foundation and the Pew and McKnight Foundations.

Journal Reference:

1. Laure Verret, Edward O. Mann, Giao B. Hang, Albert M.I. Barth, Inma Cobos, Kaitlyn Ho, Nino Devidze, Eliezer Masliah, Anatol C. Kreitzer, Istvan Mody, Lennart Mucke, Jorge J. Palop. Inhibitory Interneuron Deficit Links Altered Network Activity and Cognitive Dysfunction in Alzheimer Model. Cell, 2012; 149 (3): 708 DOI: 10.1016/j.cell.2012.02.046

Courtesy: ScienceDaily

'Rogue DNA' Plays Key Role in Heart Failure, Study Shows

DNA from the heart's own cells plays a role in heart failure by mistakenly activating the body's immune system, researchers have found. Scientists from King's College London and Osaka University Medical School in Japan showed that during heart failure -- a debilitating condition affecting 750,000 people in the UK -- this 'rogue DNA' can kick start the body's natural response to infection, contributing to the process of heart failure.

During heart failure immune cells invade the heart, a process called inflammation. The process makes heart muscle less efficient, reducing its ability to pump blood around the body. Inflammation is usually only activated when the body is facing a threat, such as an infection by a bacteria or virus.

The study, published in the journal Nature, shows in mice that inflammation in the heart can be caused by the body's own DNA. The DNA escapes when a natural process to break down damaged cell components, called autophagy, becomes less efficient. Autophagy can stop working correctly when cells are under stress, such as during heart failure.

The problem DNA comes from energy-generating structures in heart cells, called mitochondria. Mitochondrial DNA triggers inflammation because it resembles DNA from bacteria, triggering a receptor in immune cells called Toll-like Receptor 9 (TLR9).

Mitochondria fascinate scientists because they seem to have evolved from bacteria more than 1.5 billion years ago, when primitive forms of life recruited bacteria to help them produce their energy. Although this pact with bacteria is one of evolution's success stories, this study shows that the human immune system still recognises the bacterial fingerprint in mitochondrial DNA, triggering a response from the immune system.

Professor Kinya Otsu, recently announced as BHF Professor of Cardiology at King's College London, who led the study, said: 'When mitochondria are damaged by stress, such as during heart failure, they become a problem because their DNA still retains an ancient bacterial fingerprint that mobilises the body's defences.

'We previously showed that damaged mitochondria build-up during heart failure, when the natural processes of cell breakdown become less effective. Now we've shown that the DNA fingerprint that we retain in our mitochondria causes our own immune system to turn against us.'

Dr Shannon Amoils, Research Advisor at the BHF, said: 'This intriguing discovery is an important breakthrough in our understanding of why, during heart failure, the immune system becomes activated without the presence of any obvious external threat. This inflammation in the heart plays an important role in the disease process.

'Heart cells are packed with mitochondria, which provide the power the heart needs to pump blood around the body, and this study shows that, during heart failure, DNA from these mitochondria at least partly causes the problem. This research points towards new avenues of exploration that could hopefully lead to treatments for heart failure in the future.'

Professor Kinya Otsu was recently awarded more than £3 million by the BHF to carry out his pioneering work.

Journal Reference:

1. Takafumi Oka, Shungo Hikoso, Osamu Yamaguchi, Manabu Taneike, Toshihiro Takeda, Takahito Tamai, Jota Oyabu, Tomokazu Murakawa, Hiroyuki Nakayama, Kazuhiko Nishida, Shizuo Akira, Akitsugu Yamamoto, Issei Komuro, Kinya Otsu. Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure. Nature, 2012; DOI: 10.1038/nature10992

Courtesy: ScienceDaily